Facile detection and quantitation of particular nucleic acid sequences in biological samples using various methodologies offers the healthcare field new research and diagnostic capabilities that extend from the identification of the underlying genetic basis of disease to identifying pathogens and monitoring the effectiveness of therapies to both infectious and noninfectious diseases. As new nucleic acid targets for measurement are identified and correlated with particular dysfunctions, increasing need has evolved in the sensitivity and accuracy of the detection methodologies, as well as the ability to perform assays rapidly and automatedly. Most detection methodologies employ nucleic acid amplification procedures such as PCR, with various means for detecting particular amplified sequences that may be present in a sample, such as the use of labeled, hybridizable probes. PCR may be performed in various formats, such as competitive and real time. Of particular note is the combination of real-time PCR with one or more fluorescent probes referred to as molecular beacons, which fluoresce at a particular wavelength only when hybridized to a particular target sequence, which may differ from another, unrecognized sequence by only a single nucleotide, as described by Tyagi et al., 1996, Nature Biotechnology 14:303-8; European Patent Application EP 745690; and International Patent Application WO 98/10096, incorporated herein by reference.
One application of the methodologies described above is in determining the abundance of one or more particular nucleic acid sequences in a cellular sample, and in particular, the abundance of the particular sequence(s) on a per cell basis in the sample. In order to determine the number of cells from which the nucleic acid sample is derived, various procedures have been used. These include such burdensome methods as counting the number of cells prior to the preparation of nucleic acid from the cells, or using a more readily measurable marker of the number of cells from which the sample is derived. For example, the amount of DNA per cell may be determined, then the amount of DNA in a sample measured and extrapolated to the number of source cells. This procedure is inaccurate and moreover, burdensome, as these and other procedures require the concurrent determination of cell number utilizing a different procedure than the nucleic acid quantification procedure also to be applied to the sample. Methods have been developed in which the number of input cells have been determined by measuring a particular genomic nucleic acid sequence in the sample, such as that of the .beta.-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. However, the number of copies of the genes for these proteins is unknown and pseudogenes exist, and as such the denotation of the number of copies of the target nucleic acid per genome is not on a per genome basis and thus, may not be accurate.
As mentioned above, the need for accurately determining the number of copies of a particular nucleic acid sequence per cell in a biological sample has important utility in uncovering relationships between nucleic acid sequence abundances and dysfunctions, as well as diagnosing and monitoring therapies. Such sequences may be genomic or extrachromosomal, including viral, microbial or cellular. For example, a diagnostically important value is the number of infectious particles per cell, such as viruses and proviruses (HIV-1, Kaposi's sarcoma virus [human herpesvirus 8], hepatitis B and C, for example), bacteria (mycobacteria, for example), fungi, and parasites (malaria and Leishmania, for example). Other diagnostically important assessments include non-infectious agents. One particular utility is in the assessment of thymic function. The thymus is a lymphoid organ serving as the site for T cell differentiation, enlarging during infancy, stabilizing until puberty, then declining in size, and believed until recently, in function, after the third decade. Measurement of thymic function is an indicator of the ability of the immune system to recover from or become reconstituted after therapies which destroy immune cells, such as chemotherapy or radiotherapy, and to monitor the course of diseases and therapies directed thereto in infectious and noninfectious diseases involving the immune system, such as HIV-1 infection, congenital immunodeficiency disorders, as well as iatrogenically-induced immunodeficient states, for example, by radiotherapy or chemotherapy for the treatment of dysproliferative diseases. The numbers of lymphocytes in the blood that are of recent thymic origin is a measure of the output of newly generated T cells from the thymus. The numbers lymphocytes in the blood may be determined by detecting in nucleic acid isolated from peripheral blood mononuclear cells (PBMCs) the abundance of a particular species of extrachromosomal DNA formed during the excisional rearrangement of T cell receptor (TCR) genes. In the formation of .alpha..beta. and .gamma..delta. cells (bearing unique TCRs), excisional circles called a deletion circles and .delta. deletion circles, respectively, are formed (von Schwedler et al., 1990, Nature 345:452-6). As shown recently by Douek et al. (1998, Nature 396:690-5), measurement of a circles in normal subjects from birth to 73 years of age using quantitative competitive PCR showed sustained output of T cells from the thymus. Furthermore, HIV-1-infected individuals showed suppressed thymic function, but after undergoing highly active antiretroviral therapy, showed a rise in the number of a circles in CD4+ cells. These studies established the value in monitoring thymic function using TCR gene deletion circles, without precisely determining the abundance of such circles on a per cell basis. Thus, an important addition to the knowledge of an HIV-1 patient's viral load and CD4+ cell count would be thymic function as described above.
Other conditions in which immune recovery or reconstitution is an important parameter for monitoring therapies, such as bone marrow transplant, for congenital immunodeficiency disorders, such as DiGeorge syndrome, characterized by absence or hypoplasia of the thymus and a partial or complete T cell (but not B cell) deficiency. Furthermore, recovery from induced immunodeficiencies, for example, cancer treatment, using radiation and/or chemotherapeutic agents, may be monitored by assessing recent thymic emigrants. To follow individual patients, determine precise relationships between stages of disease and thymic function, and to establish normal and abnormal ranges, normalization of TCR circles on a per cell basis is necessary.
The abundance of other nucleic acid sequences expressed on a per cell basis is also important for following the status of individual patients, determining precise relationships between stages of disease and recover with particular sequences, and to establish normal and abnormal ranges. Thus, both longitudinal and cross-sectional studies of individuals may be performed to monitor individuals and establish correlations.
A further utility of genetic sequences of a known number of copies per cell is in the determination of cell numbers from biological samples, particularly when assessment methods involving gene amplification or other genetic methods are used for the analysis. For example, determining the number of cells such as leukocytes or germ cells such as sperm in a bodily fluid sample or the number of cells in a tissue sample may be determined by nucleic acid assessment methods by determining the number of copies of a particular genetic sequence known to exist at a fixed number of copies per cell.
It is thus toward a method for identifying genetic sequences suitable for use as genomic equivalent markers present at a particular copy number per genome, and the utility of the marker in determining numbers of cells and the number of copies per cell of a particular nucleic acid sequence that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.